Project Summery In the optic nerve, oligodendrocytes (OLs) are sole myelin-producing cells. Myelin provides insulation and trophic support for RGC axons and allows for normal vision. Death of OLs and demyelination in the optic nerve are the hallmarks of demyelinating diseases that often impair vision, including optic neuritis and multiple sclerosis. The signaling mechanisms that controls OL survival and demyelination are still poorly understood. Previous studies have identified a myriad of extracellular cues and OL cell surface receptors that mediate OL survival and differentiation but the intrinsic pathways that link these trophic cues to downstream events remain elusive. In my preliminary studies, I made a striking discovery by identifying Transcription Factor EB (TFEB) as the missing link. I discovered that TFEB is highly expressed by OL lineage cells in the CNS including the optic nerve. I generated a novel TFEB conditional mouse line and showed that in the mouse brain TFEB powerfully antagonizes myelination by specifically promoting premyelinating OL cell apoptosis and simultaneously inhibiting OL maturation. I developed a live-imaging based assay to model in vivo OL development, and demonstrated that TFEB directly regulates gene expression of the integrated stress response and GPCR-PKA pathways, two critical pathways previously implicated in demyelinating diseases. Based on these findings, I propose to test the hypotheses that TFEB serves as the critical sensor in premyelinating OLs that facilitates cell apoptosis in the absence of axonal contact or trophic support, and that extracellular signals modulate TFEB expression/activity through the GPCR-PKA axis to control OL maturation, myelination, and remyelination. I will utilize the rodent optic nerve as a model system to test these hypotheses. In my K99 phase I will investigate TFEB function in optic nerve myelination during development and its roles in remyelination in optic neuritis animal models, utilizing cell-type specific TFEB conditional mutants that I have already generated. I will perform whole-genome RNA sequencing experiments to identify the candidate pathways that TFEB regulates, and will further validate these pathways with the optimized in vitro culture system and the in vivo viral manipulation toolkits. To assess the functional efficacy of optic nerve remyelination and repair, I will acquire expertise in animal visual behaviors and stereotaxic injection techniques from the mentoring labs. Finally, as an independent investigator, I will employ unbiased genomic and biochemical approaches to identify the direct gene targets and interacting proteins of TFEB. I will leverage K99 phase training in animal surgery techniques and viral approaches to modulate TFEB function in promoting myelin repair, as well as the training in mouse visual behaviors to assess visual function recovery in optic nerve remyelination. The proposed research will characterize a novel pathway regulating optic nerve myelination and will determine the underlying mechanisms. By developing new drugs that target the TFEB pathway, my goal is to lessen optic nerve damage and promote remyelination and visual recovery in patients with optic neuritis and multiple sclerosis. !